Reticulocyte assay control

ABSTRACT

A pre-determined concentration of stabilized, maturation-arrested porcine reticulocytes in a red blood cell base, useful as a reticulocyte control composition. The composition can be provided in the form of a concentrated reticulocyte composition, to be diluted to a desired final reticulocyte concentration at the time of use. The composition can also be provided in the form of a diluted, ready-to-use control composition. Also included is a method of preparing such a composition, the method involving sequential steps of forming and sedimenting Rouleaux bodies.

This application is a continuation of application Ser. No. 09/055,371,filed Apr. 6, 1998, now U.S. Pat. No. 5,858,789, which is a continuationof U.S. application Ser. No. 08/695,515, filed Aug. 12, 1996, now U.S.Pat. No. 5,736,402, which is a continuation of application Ser. No.08/321,801, filed Oct. 12, 1994, now abandoned.

TECHNICAL FIELD

The present invention relates to the field of hematology, and inparticular, to assays for determining the number or presence ofreticulocytes in blood. In another aspect, the invention relates tocontrol compositions for use in performing such assays, and to processesfor preparing and methods of using such control compositions.

BACKGROUND OF THE INVENTION

The field of hematology involves the study of blood, including thediscrete cell type populations that make up the blood. One importanttool in this field involves the ability to reliably and accuratelydistinguish and count the various cell type populations. Clinicalsignificance can be attributed to abnormal levels, in both relative andabsolute terms, of most, if not all cell type populations.

In particular, the study of the kinetics of blood cell production anddestruction depends on an assessment of the number of new cells beingdelivered to the circulation per unit time. New red cells (known asreticulocytes) are easily identified and can be quantified as apercentage of total red blood cells ("RBC's"). This percentage can bemultiplied by the red cell count in order to provide the number permicroliter.

In order to identify reticulocytes, the cells can be distinguished frommature red blood cells by the presence in reticulocytes of a distinctivestainable marker formed of mitochondria, ribosomes, and othercytoplasmic organelles. This marker is visible when precipitated byso-called supravital dyes, such as new methylene blue, brilliant cresylblue, and acridine orange.

Reticulocytes require on the order of 3 to 4 days for maturation intomature (non-stainable) red blood cells, with about 2 to 3 days of thisperiod being spent in the marrow itself, and about one day in theperipheral blood. Since approximately 1% of the circulating red cells innormal humans are replaced daily, and a newly released erytlirocyte isidentifiable as a reticulocyte for only about one day, it follows thatunder normal circumstances about 1% of circulating red cells aredetectable as reticulocytes.

Variations in the reticulocyte count can be clinically significant, ifdetected with assurance. Levels approaching 3% (based on the totalnumber of circulating red cells) are seen in instances of increasedmarrow activity, e.g., when blood synthesis is stimulated as byerythropoietin. In contrast, levels below about 0.5% can be anindication of bone marrow incompetence. Hence, a reticulocyte count isan effective measure of marrow erythroid output, since the release ofone reticulocyte signals the production of one red cell by the marrow.

A wide variety of approaches have been described for determiningabsolute and/or relative reticulocyte levels. Although such approacheshave traditionally been performed manually, automated procedures arebeing used with increasing frequency. An increasing number of majorinstrument manufacturers of automated cell counting instruments haveadded the ability to count reticulocytes to their systems in recentyears. Some instruments provide values for what is known as a"reticulocyte maturation index", or "RMI". Although this parameter isnot yet standardized for clinical reporting, it nevertheless shows greatpromise as a clinically useful parameter.

A common principle underlying the use of each method is the need for areliable, standardized reticulocyte control composition, against whichto assess the reliability of the results. Presently, reticulocytecontrols are provided in a number of different forms. Human blood itselfis generally considered to be unsuitable for the preparation ofreticulocyte controls. Reasons for this include the safety concernsassociated in recent years with the use of human blood products, as wellas the fact that human blood is typically too low in reticulocyte countto be useful for the preparation of a wide range of control levels.

A control composition known as "Retic-C™", is available in controllevels I, II and III from Coulter Corporation (Miami, Fla.). Thisproduct is includes avian red blood cells as the reticulocyte analogues.Such cells are not derived from true reticulocytes, however, nor do theystain in the manner common to reticulocytes. As a result, thecomposition is limited in use to automated instruments that rely ondetection techniques other than traditional staining, such as theCoulter "STK-S™" instrument. The avian cells are significantly largerthan human reticulocytes and, in contrast to reticulocytes, also containa nucleus.

Another control composition is a reticulocyte analogue product availableas the "Retic-CHEX™" product in concentrations of 1, 3, and 5% fromStreck Laboratories (Omaha, Nebr.). This control also suffers from anumber of drawbacks, however, including poor staining intensity, andperformance that is only minimally like that of true reticulocytes. Yetother control compositions, both of which are manufactured by Streck,include "Test Point™" product (available in 2 and 5% levels for use withMiles/Technicon instruments), and a 2 and 5% level control available forinstruments available from Sysmex. Both compositions are limited in thatthey provide only two levels, with the upper level being significantlylower than may be desired.

As a result, there do not appear to be any control compositionsavailable on the market that provide an optimal combination ofstability, true reticulocyte appearance and stain characteristics, andwide utility with most, if not all, presently available assaytechniques. A reticulocyte control product capable of providing suchattributes would be highly desirable.

SUMMARY OF THE INVENTION

The present invention provides a reticulocyte control compositioncomprising a predetermined concentration of stabilized,maturation-arrested porcine reticulocytes in a red blood cell base. Inone preferred embodiment, the composition is in the form of aconcentrated reticulocyte composition, having on the order of about 85%or more porcine reticulocytes (based on the total red blood cell count).The concentrate can be combined with a diluent porcine or human redblood cell base, e.g., having a similar total cell count, but on theorder of 1% reticulocytes. The concentrate and diluent can be combinedat the time of use, in order to achieve a desired final reticulocyteconcentration.

In an alternative preferred embodiment, the concentrate composition canbe included in a concentrate/diluent kit, together with one or morediluent RBC base compositions, for mixing at the time of use. In yetanother preferred embodiment, a plurality of compositions are providedin dilute, ready-to-use kit form, the kit having a plurality ofpredetermined concentrations of reticulocytes. Control compositions ofthe present invention provide an optimal combination of flexibility,concentration range, uniformity and shelf-life.

In another aspect, the invention provides a method of preparing areticulocyte control composition, the method including the steps of: (1)harvesting an enriched population of porcine reticulocytes; (2)arresting further maturation of the reticulocytes and stabilizing theharvested, arrested reticulocytes; and (3) preparing a controlcomposition comprising the stabilized, maturation-arrestedreticulocytes.

A preferred method of the invention involves the initial step ofpreparing a fraction of porcine blood that is enriched for the presenceof reticulocytes. In a particularly preferred embodiment, the method ofthe present invention is integrated with present-day preparativeprocedures involving other uses of porcine blood, e.g., the recovery ofporcine platelets.

Applicants have found that existing procedures for the recovery ofporcine platelets can also be used to provide fractions that areenriched in the presence of reticulocytes. Such fractions provide auseful and cost-effective starting point for the preparation ofreticulocyte control compositions. The resulting control compositionsprovide an optimal combination of stability, true reticulocyteappearance and stain characteristics, and wide utility as a control withmost, if not all, presently available assay techniques.

DETAILED DESCRIPTION

Applicants have observed that porcine blood provides a naturally higherlevel of reticulocytes, as compared to humans, and discovered that thesereticulocytes can be recovered and stabilized in a manner that rendersthem useful as a reticulocyte control composition. Porcine reticulocytesappear to enter the circulation earlier, and in turn tend to be youngerin appearance than counterpart cells in human blood. As such, theyprovide an opportunity to recover a substantially homogeneous populationof cells that can then be arrested to minimize further development, andprovided in arrested, stabilized form.

A preferred method involves sequential sedimentation steps, both ofwhich involve the formation and sedimentation of "Rouleaux bodies", asdescribed below. In the first step, a platelet-rich supernatant andcorresponding reticulocyte-rich sediment fraction are formed. Theplatelet rich supernatant can be removed and used for other purposes,while the sediment fraction can be resuspended and subjected to a secondsedimentation step. The supernatant fraction formed as the result of thesecond sedimentation step has been found to be particularly rich inreticulocytes. The second sediment fraction, in turn, has been found tobe particularly useful as a diluent base.

In a preferred embodiment, the reticulocyte-rich second supernatantfraction contains on the order of 80% to 95% or more reticulocytes (as apercentage of the total RBC's per unit volume). Correspondingly, thesecond sediment fraction contains on the order of 0.5% to 1%reticulocytes. Accordingly, desirable control compositions having anylevel between 0.5% and 95% or more can be prepared by judiciouscombination of the second supernatant and sediment (diluent) fractions.

Applicants have found that dextran-induced Rouleaux formation can beused to provide an efficient separation of reticulocytes and mature redblood cells for the purpose of the present invention. The ability tocontrol the manner and extent to which Rouleaux bodies will sedimentwith and without reticulocytes serves as the basis for the uniquetwo-step sedimentation protocol of this preferred method.

Reticulocytes are immature red blood cells. In both human and porcineblood, the reticulocytes tend to be larger and less concave than theircorresponding mature red blood cells. Reticulocytes also tend to possesshigher surface charge densities than the corresponding fully maturecells. As a result, at least in porcine blood, reticulocytes do notgenerally form Rouleaux bodies, either with each other or with maturered blood cells. Under suitable circumstances, however, Applicants havefound that porcine reticulocytes can nevertheless be caused to sedimentwith Rouleaux bodies.

In contrast, Applicants have discovered that under certain conditionshuman reticulocytes can form Rouleaux bodies in the presence of maturehuman red blood cells. As a result, the method described in the presentinvention finds greater utility with porcine reticulocytes, which aremuch easier to separate from the mature porcine red cell population.

Additionally, pigs normally possess approximately twice as manyreticulocytes (as a percentage of total red blood cells per unit volume)in comparison to humans. Applicants have found that the relativeabundance of reticulocytes, the large volumes of blood that areavailable, and the strong discrimination against reticulocyte Rouleauxformation in the pig, can all be used to effectively produce largevolumes of an enriched fraction having a high reticulocyteconcentration.

As used in this application the term "reticulocyte control" will referto a suspension of porcine cells useful as a control in one or moremanual or instrument-based assays for determining the number or level ofnatural, e.g., human, reticulocyte cells. The word "arrest", andinflections thereof, will refer to porcine reticulocytes that aresubstantially inhibited, either reversibly or irreversibly, fromundergoing further natural maturation in a manner that would diminishtheir utility as a reticulocyte control. Typically, maturation ofreticulocytes corresponds with a loss of distinctive cellular materialthat is stainable with conventional chromogenic or fluorogenic paravitaldyes.

The word "stabilize", and inflections thereof, as used herein, willrefer to arrested cells that are provided in a form that allows thecells to be stored and used in a manner that does not unduly diminishtheir utility as a reticulocyte control. Generally, stabilizationinvolves the preparation of a suspension containing such cells incombination with one or more ingredients that are useful aspreservatives. In a preferred embodiment, reticulocyte controlcompositions are provided in the form of concentrated suspensions having2 month, and preferably 3 month or longer shelf stability when storedunder refrigeration.

Compositions of the present invention can be prepared from whole porcineblood, or fractions thereof, according to any suitable preparativeprocedure. The following is a general description of a preferred methodfor the harvest of porcine reticulocytes and preparation of areticulocyte control composition having a plurality, e.g., three,concentration levels.

Porcine blood can be recovered from any suitable source. Preferredprocedures for the preparation of platelets involve the recovery ofporcine blood from freshly slaughtered pigs. One particular advantage ofthe preferred method of the present invention is that blood can be usedfor the recovery of reticulocytes in a manner that is compatible withthe use of the blood for other purposes, e.g., the recovery ofplatelets.

U.S. Pat. Nos. 4,324,686 and 4,338,564, the disclosures of which areincorporated herein by reference, describe methods for the preparationof compositions and controls containing porcine blood platelets. Themethods described in these patents are particularly preferred for use inthe initial steps of the presently claimed method.

In one embodiment of the method of the invention, a first sedimentationstep is employed in order to provide a platelet-rich supernatant, and areticulocyte-rich sediment fraction. The method involves the use of areagent such as a citrate-citric acid-dextran ("CCD") reagent, which isadded to whole blood to facilitate the separation of a red blood cellsediment or pellet, and a platelet-rich plasma supernatant.

Newly recovered blood is added directly to a fill large (e.g., 20 liter)carboy containing a suitable amount of the dextran-based anticoagulantconcentrate. A suitable formulation for the preparation of ananticoagulant/dextran concentrate is provided below. Given the teachingof the present invention, those skilled in the art will be able todetermine the actual amount of the anticoagulant/dextran concentratethat is sufficient to prevent blood clot formation in the blood sample,while at the same time allow Rouleaux formation of red blood cells asdescribed below. The dextran has been a useful component in thecollection of porcine blood for the purpose of harvesting platelets fromthe fresh porcine blood.

The Rouleaux effect can be assessed by microscopic evaluation of asample of blood. The effect is typified by a distinctive stacking oraggregating of mature red blood cells together in rows. Mature red bloodcells, both porcine and human, are bi-concave in shape and have arelatively low surface chemical charge. These properties allow them toform the Rouleaux bodies in the presence of a high molecular weightpolysaccharides (e.g., dextran) or polypeptides (e.g., gelatin).

The mixture of whole blood and CCD concentrate is prepared and allowedto settle at room temperature for one to three hours. After settling, adense sediment is formed that is dark red in appearance. Above thesediment there is a translucent pink-colored, platelet-rich supernatant.The supernatant (generally representing about 12 liters of the original20 liters) is removed, as by aspiration or gentle pouring, taking carenot to disturb the interface.

Applicants have discovered that the sediment is particularly rich inreticulocytes, even though reticulocytes are not believed to themselvesform Rouleaux bodies with mature RBC's. Rather, it appears that thereticulocytes are trapped within or between, but do not actually form apart of, the Rouleaux bodies formed by the pig RBC's. In order torecover the reticulocytes, this first sediment is resuspended to theoriginal sample volume by the addition of diluted CCD. The diluted CCDis added to achieve or maintain a final concentration of CCD equivalentto the concentration achieved in the first mixture of blood and CCDconcentrate.

The resuspended first sediment is incubated overnight (e.g., about 15hours) at room temperature, in order to again allow the formation andsettling of Rouleaux bodies. Upon incubation there again appears a lowercompact cell sediment, and an upper supernatant fraction that is darkerred in appearance than the first supernatant. The two fractions areseparated, washed, and assayed in order to obtain concentrated anddiluent reticulocyte phases.

The supernatant of the second sedimentation has been found to beparticularly rich in reticulocytes. Upon removal from the secondsediment, the supernatant is preferably filtered by any suitable means,in order to remove white blood cells and platelet aggregates. In thepreferred filter method described below, the supernatant is filteredthrough a cellulose slurry filter. White blood cells ("WBC") andplatelets are retained by the slurry, while the filtrate is collectedand assayed to ensure that the WBC and platelet levels are withindesired specifications. Typical specifications include 2,000 or lessWBC's, and 40,000 or less platelets, per microliter.

The filtered reticulocyte-containing suspension is then centrifuged toconcentrate the cells, which are then resuspended in a liquid mediumdesigned to stabilize the cells and substantially arrest furtherreticulocyte maturation.

The second sediment fraction is also recovered, then washed one or moretimes to remove WBC's and platelets, and finally resuspended to adesired extent to serve as a low-reticulocyte diluent.

Preferably in the course of the above process the enriched porcinereticulocytes are arrested from further maturation and stabilized. Theinhibition of maturation and the stabilization of the cells can beaccomplished by resuspending the cells in a suitable medium. Applicantshave discovered that media such as those described previously for therecovery of pig platelets can be useful for preparing a reticulocytecomposition as well. As a result, the method and composition of thepresent invention can be integrated with platelet recovery methodologiesin order provide an optimal and cost effective use of the blood.

A medium useful for the recovery and storage of porcine reticulocytes,which will be referred to for brevity as a "Retic Medium", is providedin the form of a buffered solution maintained at a pH between pH 7 andpH 8. The solution preferably includes a protein synthesis inhibitor orother metabolic inhibitor capable of arresting mammalian red celldevelopment. Preferred inhibitors include natural antibiotics such ascycloheximide, and semisynthetic antibiotics such as rifampin, each usedat a final concentration of about 0.25 to 1% (by weight, based on thevolume of the medium).

The Retic Medium also preferably lacks a carbon or other energy sourceuseful for continued metabolism. In the absence of maturation inhibitorsand/or in the presence of an energy source, reticulocytes may continueto develop and lose their stainable RNA material, even at lowertemperatures.

Cycloheximide is particularly preferred, since it appears to provide anirreversible stabilizing effect on the porcine reticulocytes. Attemptsto induce maturation in porcine reticulocytes after brief exposure toRetic Medium containing cycloheximide have resulted in reticulocytesthat do not lose their stainable RNA/protein material. Thus, with regardto the detectable marker for reticulocyte enumeration (cytosolicribonucleoprotein), the porcine reticulocytes appear to be stabilized.

More preferably, the solution includes one or more preservatives, inorder to provide the final composition with prolonged shelf lifestability. For example, fairly young reticulocytes in human blood cellsamples were found to be stable after storage for over 2 months at 4° C.in a Retic Medium such as exemplified below. Suitable preservativesinclude, but are not limited to, nalidixic acid (an antibacterialagent); p-hydroxybenzoate methyl ester and p-hydroxybenzoate propylester (antibacterial agents); and penicillin (antibacterial).

A control composition of the present invention can be used according toestablished laboratory hematology procedures, for instance, as a controlto calibrate or monitor the performance of diagnostic tests. Preferredcompositions are composed of stable materials that provide a means ofverifying accuracy and precision of reticulocyte counting methods, andcan be handled in the same manner as patient specimens.

A composition of the present invention is preferably provided in theform of an in vitro diagnostic reagent that includes arrested,stabilized porcine reticulocytes in combination with porcine, human, orother mammalian erythrocytes, all suspended in a plasma-like fluidcontaining suitable preservatives. Any human blood components used inthe preparation of such a composition should be suitably tested in orderto ensure that it is negative for all necessary infectious agents orindicators, including hepatitis B surface antigen (HBsAg), hepatitis Cvirus antibody (anti-HCV), and human immunodeficiency virus antibody(anti-HIV). Since no test available can provide total assurance thatspecimens of human origin will not transmit infectious disease,particular care should be used with any such composition.

In a preferred embodiment, a composition of the present invention isused to prepare a plurality of control levels, e.g., three levelsrepresenting normal, moderately high, and very high levels ofreticulocytes.

A control composition of the present invention is typically provided inthe form of a kit containing a plurality of different reticulocyteconcentrations. A typical kit includes separate vials containingreticulocytes at a plurality of concentrations ranging between about0.5% to about 25%. Most preferably, the concentrations range betweenabout 1% and 15%, and even more preferably, between about 1% and 12%. Apreferred kit, for instance, includes a plurality of concentrations,e.g., 1%, 5%, and 12%.

Concentrations over about 25% tend to have little clinical usefulness,since they are rarely encountered in practice. On the other hand,concentrations less than about 0.5%, are difficult to determine withboth accuracy and precision by conventional detection techniques.

Compositions of the present invention are particularly stable instorage, and are preferably stored upright at temperatures of between 2°and 8° C. when not in use. Compositions should be protected fromoverheating and freezing. Unopened vials containing such compositionsare stable until the expiration date, while opened vials are stable forat least 14 days provided they are handled properly. A prepared sampleof a composition of this invention is stable for on the order of 60 to90 minutes.

Diluted, ready-to-use compositions of the invention are generallysimilar in appearance to fresh whole blood. It is normal for a lightpink-tinted supematant to form upon settling. Discoloration of thesupernatant fluid or visible hemolysis may indicate productdeterioration. Overheating, freezing, rough handling, and contaminationare frequent causes of product damage. Inability to recover expectedvalues may also indicate product deterioration. Incomplete mixing,instrument malfunction, or defective stains are other causes ofunacceptable results. A composition should not be used if deteriorationis suspected.

In use, a vial containing the composition of the present invention istypically removed from refrigerated storage and allowed to warm to roomtemperature. The composition is then mixed by gently rolling the vialbetween the palms of the hands, and inverting the vial until the cellsappear to be completely and uniformly suspended. Care should be taken toavoid undue agitation of the vial, such as by use of a vortex, sinceagitation can disrupt the cells.

The composition can then be incorporated into the analytical procedureof choice in the same manner as a patient sample, for instance withautomated methods, the control is analyzed in the manner provided withthe operator's manual for the particular instrument.

The composition should be used within a reasonable time, depending onthe particular method employed (e.g., within about 60 minutes, afterwarming to room temperature). After removing an aliquot from the vial,the control composition can be re-stored for later use by carefullywiping the vial rim and cap and returning the capped vial to therefrigerator.

Assay values are typically presented as a mean and a range. The mean isderived from replicate testing by the specific method. Instruments usedfor automated methods are operated and maintained according to theinstrument manufacturer's instructions. The manual method is a directmicroscopic count using the conventional reticulocyte counting procedurewith new methylene blue stain.

The range is an estimate of variation between laboratories and takesinto account inherent impression of the method, differences inmaintenance, operating technique, and equipment. It is recommended thateach laboratory establish its own laboratory-specific ranges for greatercontrol sensitivity.

Assay values on a new lot of control should be confirmed before it isput into routine use. The laboratory recovered mean should be within theassay range. Laboratories may consider results acceptable when at least95 percent of results are within 2 Standard Deviations of the laboratorymean.

A composition of the present invention can be used as a controlcomposition in most, if not all, of the following conventional methodsfor reticulocyte evaluation.

Manual (microscopic):

A suitable procedure for the manual evaluation of reticulocytes involvesthe mixing of an equal amount of blood with a supravital stain, e.g.,new methylene blue or brilliant cresyl blue, and incubating the mixturefor approximately fifteen minutes. This incubation period allows the RNAto precipitate with protein as a complex, which then appears as a darkblue network that allows reticulocytes to be identified and enumeratedmicroscopically.

The sample is mixed well and three wedge smears are prepared. In oneapproach, a technician can examine the smear and count 500 cells underthe 100× oil objective. Using a standard "bafflement" pattern, the redblood cells and reticulocytes are counted simultaneously using separatecell counters. A second technician can use the same technique on adifferent smear. The two counts must agree within 10%. If they do not, athird smear should be counted. The reticulocyte percent is calculated asfollows: ##EQU1##

Another approach involves the use of a Miller disc inserted into aneyepiece. The disc is composed of two squares. The area of the smallersquare is 1/9 that of the larger square. The red blood cells are countedin the smaller square, while the reticulocytes are counted in the largersquare. Cells should be counted in 20 successive fields. Using thisapproach, the reticulocyte percent can be calculated as follows:##EQU2##

Caution must be taken to ensure that cellular inclusions, such asHowell-Jolly bodies and Heinz bodies, are not confused with theidentification of reticulocytes. Precipitated stain and refractileartifact must also be distinguished from reticulocytes.

Control compositions of the present invention perform particularly wellin manual assays. The porcine reticulocytes are quite similar in bothappearance and performance to human reticulocytes. They also tend to beyoung and easily identifiable. Even at higher concentrations, theporcine reticulocytes provide a useful visual contrast between thereticulocytes and unstained, mature RBC's.

Flow Cytometry:

Thiazole orange has proven to be the reticulocyte fluoroindicator ofchoice when counting reticulocytes on a semi-automated flow cytometersuch as a Becton Dickinson FACScan™, Coulter EPICS or an Ortho Spectrum.Staining with thiazole orange increases with time and temperature, andthose skilled in the art will appreciate the manner in which theseparameters can be standardized. Thiazole orange is available from BectonDickinson in pre-diluted form as the product known as "ReticCOUNT™".

Yet another fluorescent indicator, known as "auramine o" is currentlyused on reticulocyte automated flow cytometers available from Sysmex,but has not found favor in the semi-automated cytometers. A recentlydeveloped fluorochrome that may find use for flow cytometricreticulocyte counting is known as "Syto 12™" from Molecular Probes ofEugene, Ore. This dye is one of several new supravital stains thatpermeate cells rapidly and produce high fluorescent yields upon bindingto nucleic acids.

SYSMEX R-1000™ AND R-3000™:

In 1989, TOA Medical Electronics Co. LTD, Hobe Japan, introduced abenchtop flow cytometer known as the "Sysmex R-1000™" automatedreticulocyte analyzer. Three years later, an upgraded R-series analyzer,the R3000™ was released. The blood sample is aspirated, diluted andstained automatically by both instruments. The supravital dye used isauramine o, which carries a positive charge, resulting in an attractionto the negatively charged ribonucleic acid (RNA) and deoxyribonucleicacid (DNA). The samples are hydrodynamically focused and passed througha flow cell. The cell is illuminated by an argon laser beam at awavelength of 488 nm.

The R-3000 measures forward fluorescence using a filter to obstruct ashorter wavelength of light and a photomultiplier tube to determine theintensity. The R-1000 measures side fluorescence using a filter placedat a 90 degree angle to the laser beam path. The forward scattermeasures the cell size and fluorescence indicates the RNA content of thesample. The fluorescence and scattered light of the individual cellsallow these instruments to count the reticulocytes. The instrumentsanalyze about 30,000 cells per sample, determining the reticulocytecount, reticulocyte ratio, and total RBC count. This methodology cananalyze far more cells than the manual method, thus increasing thereproducibility and precision. Both instruments use scattergrams dividedinto three areas to display results. These areas are platelets, matureRBC's and reticulocytes.

The reticulocyte area is further divided into three areas; lowfluorescence ratio (LFR), middle fluorescence ratio (MFR) and highfluorescence ratio (HFR). An additional area that displays very highfluorescent events counts "shift" cells, nucleated RBCs and cellscontaining Howell-Jolly bodies. These cells are not included in thereticulocyte count. The area is found above the upper limit of mostreticulocytes and below the WBC region.

MILES DIAGNOSTICS H*3™:

Miles Diagnostics/Technicon introduced the H*3™ hematology analyzerduring 1993, partly in response to the growing demand for automatedreticulocyte counting. The H*3™ is a semi-automated flow cytometer thatanalyzes RBC's using the same technology as previously used in the H*1™and H*2™ analyzers, but also measuring hemoglobin content as well. Thevalue of measuring the hemoglobin content of reticulocytes has beendescribed in support of this unique feature of the H*3™.

Coulter STKS/MAXM:

The Coulter Corporation has developed an upgrade for their STXS and MAXMhematology analyzers. Using their VCS technology (Volume, Conductivityand Scatter), the upgrade provides reticulocyte percentage values baseon new methylene blue staining characteristics of the reticulocytes. Theprocedure requires two offline steps. First, samples are diluted into anew methylene blue solution and incubated at room temperature forbetween 5 and 60 minutes. Second, a small volume of the stained sampleis diluted with a clearing solution that clears the cellular hemoglobinbut preserves the RNA-new methylene blue complex. In addition to thereticulocyte percentage and absolute number, the mean reticulocytevolume and reticulocyte maturation index parameters are available aswell.

Control compositions of the present invention also perform quite well ineach of the automated systems described above, using methodologiessuggested by the manufacturers and within the skill of those in the art.

The invention will be further described with reference to the followingnon-limiting examples.

EXAMPLES REAGENT PREPARATION

The following reagents were prepared in advance:

CC Dextran (stock solution)

The following ingredients were combined and made to 60 liters withdeionized water: NaOH (24 g), citric acid (142.63 g), sodium citrate(3210 g), (3600 g) Dextran (Sigma No. D-4876, molecular weight range 150kd to 180 kd).

CC Dextran (diluted)

The "CC Dextran" solution was diluted 1:7.1 with standard phosphatebuffered saline (pH 7.5+/-0.1) having (0.1%) azide as a preservative("PBSN") (i.e. for 12 liters final, 1.69 liters of CC Dextran plus 10.31liters of PBSN).

Cellulose Slurry

The following ingredients were added to a 2 liter flask (each celluloseproduct was obtained from Sigma Chemical Co.):

100 g Cellulose, alpha cellulose fiber

50 g Cellulose, microgranular

50 g Cellulose (Sigmacell Type 50)

Retic Medium (to 1.5 liters)

The ingredients were thoroughly mixed and care was taken to ensure thatthe cellulose slurry was thoroughly resuspended and mixed just prior topouring into funnel beds.

    ______________________________________                                        Retic Medium                                                                  Component           Units  Qty/L                                              ______________________________________                                        Albumim, Bovine     mL     100.0                                              Chloride, Potassium g      4.0                                                Citrate, Tripotassium                                                                             g      3.0                                                Cycloheximide       g      0.5                                                Phosphate, Dipotassium                                                                            g      5.0                                                ______________________________________                                    

The ingredients were combined and the solution made to a pH of 7.5(+/-0.1), with an osmolality of approximately 340 (+/-15). After theingredients were added to the solution, sterile filtration wasaccomplished by peristaltic pumping through a terminal 0.2 micronminicapsule filter of 500 square cm surface area.

EXAMPLE 1

Porcine reticulocytes were harvested and prepared as a controlcomposition in the following manner:

1. Recovery of porcine blood.

At a slaughter house, porcine blood was recovered from freshlyslaughtered pigs and added to fill 20 liter carboys containing 2.5liters of "CCD" concentrate (prepared as described above). The finalconcentration of the CCD concentrate upon filling the carboy with freshporcine blood was determined such that blood clotting would be preventedand the red blood cells would exhibit the desired Rouleaux effect.

2. First Rouleaux sedimentation.

The blood/CCD mixture was gently transported by truck to the laboratoryand allowed to continue to settle, for a total incubation time ofapproximately 3 hours. Upon settling, a dense sediment was formed thatwas dark red in appearance. Above the sediment there was a sharpinterface between the sediment and the translucent pink-colored,platelet-rich supernatant.

3. Separation of first sediment and supernatant.

The supernatant (approximately 12 liters of the original 20 liters) wasremoved by aspiration, taking care not to disturb the interface. Theinterface (approx. 0.5 liters) was then gently removed as well, byaspiration, and discarded. The Rouleaux effect in the first sedimentfraction was confirmed by microscopic examination which revealeddistinctive mature red blood cells stacking or aggregating together inrows.

4. Dilution of first sediment, second sedimentation.

In order to recover entrapped reticulocytes, the first sediment wasresuspended to the original sample volume (20 liters) by the addition ofapproximately 12 liters of diluted CCD, prepared as described above. Theresuspended first sediment was incubated overnight (e.g., about 15hours) at room temperature, in order to allow the formation and secondsettling of Rouleaux bodies. Upon incubation there again appeared alower compact cell sediment, and an upper supernatant fraction that wasdarker red in appearance than the first supernatant.

5. Separation of second sediment and supernatant.

The upper phase was slowly aspirated to form one liter fractions, downto a level approximately 2 cm above the resedimented RBC phase. The twofractions were separated, washed, and assayed as described below inorder to obtain concentrated and diluent reticulocyte phases.

6. Processing of second sediment.

The second sediment fraction was recovered, washed to remove WBC's andplatelets, and finally resuspended to serve as a low-reticulocytediluent. The sediment was resuspended to a concentration of 3.8 to 4.2million cells per microliter, as determined by analysis on a Coulter S-4instrument. Within this suspension, it was found that between about 0.5%and 1% were identifiable as reticulocytes.

Approximately one-half of the recovered second supernatant was availablefor recovery. Optionally, where the reticulocytes are to be diluted in aporcine RBC base composition, the remaining 2 cm interface between theupper phase and sedimented phase can be aspirated and stored separately.

7. Processing of second supernatant.

Upon removal from the second sedimentation step, the supernatant wasdiluted with Retic Medium to achieve a total cell count of between 0.4and 0.6 million cells per microliter. The diluted supernatant was thenfiltered in order to remove white blood cells and platelet aggregates.The supernatant was filtered through a cellulose slurry filter using theslurry composition described above. WBC's and platelets were retained bythe slurry, while the filtrate was collected and assayed to ensure thatthe WBC and platelet levels were within specifications.

All final filtrates (reticulocyte preparations with WBC and plateletlevels within specifications) were centrifuiged at 4500 RPM in a GSArotor (RC5 Sorvall centrifuge) for 15 minutes in order to concentratethe reticulocytes.

The supernatant was poured off and the pellet resuspended in ReticMedium. The platelet level in the resuspended pellet was determined andif above specified limits, the sample was further diluted with ReticMedium and re-centrifuged in the GSA rotor at 2,000 RPM for 3 minutes.The next supernatant was gently poured off to provide a soft pellet.This process was repeated until the platelet amount was withinspecifications.

The final pellet was resuspended in Retic Medium to achieve aconcentration of 3.8 to 4.2 million cells per microliter, as determinedby analysis on a Coulter S-4 instrument. It has been found that,depending on the particular preparation, between about 80% and about 95%of the cells in this final suspension are identifiable as reticulocytes.

8. Preparation of control levels.

The final assayed fractions were used to prepare various control levels(1%, 5%, and 12%) for use in a control kit. In order to provide controlcompositions having porcine reticuloyctes in a porcine RBC base,suitable mixtures were made of the (1%) suspension formed in step 6above, with the (80-95%) suspension formed in step 7.

In order to provide control compositions having porcine reticulocytes ina human RBC base, suitable mixtures were made of the suspension formedin step 7 above with a human cell isolate prepared as described below.

Human red cells that had been isolated from blood plasma were purchasedfrom a commercial vendor of diagnostic blood. The packed cells werereceived in one-unit blood bags, and were pooled upon receipt. Thepooled blood was then further processed in repetitive steps in order toremove non-RBC components.

The pooled samples were centrifuged for 20 minutes at 2500 RPM. Whitecell material was removed from the top of the resultant soft cellpellet. The pellet was repeatedly washed until the white blood cell andplatelet concentrations in the pellet were within desiredspecifications. The final washed cell pellet was then resuspended to aconcentration of 3.8 to 4.2 million cells per microliter and used as thelower diluent and a human RBC base.

EXAMPLE 2

Concentrated and dilute reticulocyte compositions prepared as describedin Example 1 where combined at various proportions and used intriplicate to determine the correlation between a standard flowcytometric technique (EPICS) and various manual and automatedreticulocyte determination techniques. The results can be seen in TABLES1 and 2 below. The data shows that the correlation between the EPICStechnique and the various other techniques is consistently very close.When properly constructed, the data derived with the presentreticulocyte compositions can provide accurate and useful values for RMIas well. The data indicates that the reticulocyte preparations of thepresent invention have wide applicability in most, if not all, presentlyavailable instruments and techniques.

                  TABLE 1                                                         ______________________________________                                        Correlation Data                                                              Technicon H*3 ™ vs.                                                                     Coulter STKS ™ vs.                                                                       Sysmex R1000 ™ vs.                              EPICS ™ Profile II                                                                      EPICS ™ Profile II                                                                       EPICS ™ Profile II                              H*3    EPICS     STKS     EPICS  R1000  EPICS                                 ______________________________________                                        0.7    0.6       0.8      0.8    0.4    0.8                                   0.7    0.7       1.3      1.0    0.4    0.8                                   4.5    4.3       3.6      3.6    0.6    0.5                                   4.6    4.5       3.6      3.3    0.7    0.9                                   5.9    5.7       5.5      5.6    0.7    0.7                                   6.9    6.8       5.6      7.0    4.7    4.4                                   8.1    8.0       7.9      6.5    5.0    5.8                                   8.1    8.9       8.9      8.1    5.0    5.8                                   10.4   11.5      18.4     17.2   5.2    5.7                                   11.7   12.0      25.6     25.6   5.3    5.2                                   13.3   14.4      35.4     33.9   9.5    8.5                                   13.5   12.6                      10.6   11.6                                  16.0   16.0                      10.8   11.7                                  16.1   16.3                      11.0   12.2                                  26.1   27.9                      11.6   11.7                                  26.4   26.3                                                                   28.4   31.0                                                                   33.9   35.5                                                                   40.4   42.9                                                                   54.7   57.7                                                                   63.8   66.9                                                                   ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Correlation Data                                                              NMB Manual vs      Sysmex R3000 vs                                            EPICS Profile II   EPICS Profile II                                           NMB      EPICS         R3000   EPICS                                          ______________________________________                                        0.4      0.8           0.5     0.7                                            0.6      0.7           0.6     0.7                                            0.6      0.8           0.7     0.8                                            0.8      0.8           4.8     5.4                                            6.0      6.4           4.9     5.4                                            6.2      6.8           5.0     5.4                                            6.8      7.4           5.2     5.5                                            7.8      7.0           5.4     5.5                                            8.0      7.6           5.4     5.4                                            8.2      7.3           9.7     10.7                                           12.0     12.9          10.0    10.9                                           12.6     12.9          10.2    10.9                                           13.8     13.5          10.3    11.0                                           14.2     14.7          10.6    11.0                                           15.6     16.1          10.8    11.1                                           ______________________________________                                    

We claim:
 1. A system for analyzing blood to determine the population ofreticulocytes, the system comprising:a hematology instrument that iscapable of measuring a population of reticulocytes; and a reticulocytecontrol composition for use in the hematology instrument, thereticulocyte control composition comprising stabilzed,maturation-arrested porcine reticulocytes in a red blood cell base,wherein the red blood cell base comprises detectable, matureerythrocytes.
 2. A system according to claim 1 wherein the reticulocytecontrol composition is in the form of a concentrated porcinereticulocyte composition, having on the order of about 80% or moreporcine reticulocytes, based on the total detectable red blood cellcount.
 3. A system according to claim 1 wherein the compositiondemonstrates long term refrigerated storage stability of at least twomonths.
 4. A system according to claim 1 wherein the maturation of thereticulocytes has been arrested by incorporation of a protein synthesisor metabolic inhibitor.
 5. A system according to claim 4 wherein theinhibitor is selected from the group consisting of cycloheximide andrifampicin.
 6. A method of determining the accuracy of a hematologyinstrument, the method comprising the steps of:a) providing areticulocyte control composition comprising stabilized,maturation-arrested porcine reticulocytes in a red blood cell base,wherein the red blood cell base comprises detectable, matureerythrocytes; b) passing the control composition through the hematologyinstrument to measure the amount of reticulocytes; and c) comparing theamount of reticulocytes measured in step b) with assayed values of thereticulocyte control composition.
 7. A method according to claim 6wherein the reticulocyte control composition is in the form of aconcentrated porcine reticulocyte composition, having on the order ofabout 80% or more porcine reticulocytes, based on the total detectablered blood cell count.
 8. A method according to claim 6 wherein thecomposition demonstrates long term refrigerated storage stability of atleast two months.
 9. A method according to claim 6 wherein thematuration of the reticulocytes has been arrested by incorporation of aprotein synthesis or metabolic inhibitor.
 10. A method according toclaim 9 wherein the inhibitor is selected from the group consisting ofcycloheximide and rifampicin.